The present invention concerns cross-linked hyaluronic acids, optionally hemisuccinylated or sulphated, the salts thereof with biologically suitable or pharmacologically active cations and the complexes thereof with heavy metals such as copper, zinc and iron.
The invention also concerns the use of said cross-linked hyaluronic acids, salts and complexes in the medical, pharmaceutical and cosmetic fields.
Hyaluronic acid is a glycosaminoglycan consisting of disaccharide units of D-glucuronic acid and N-acetylglucosamino-2-acetamido-2-deoxy-D-glucose, connected by xcex2 (1xe2x86x923) glycoside bonds.
Natural hyaluronic acid has linear, not cross-linked structure of molecular weight ranging from 50,000 to 8,000,000 D or more, depending on the source and extraction method.
Hyaluronic acid is present in the synovial liquid, connective tissue and vitreous humor of higher animals, as well as in some bacteria.
Compositions of sodium hyaluronate having various molecular weights (in the form of solutions having different viscosities, gels with different viscoelastic characteristics, sponges, films or membranes) are used in human medicine and surgery for instance as substitutes of synovial liquid, tissular antiadhesive agents, substitutes of vitreous humor, artificial tears, agents for the in vivo tissular re-constitution (for instance as extra-cellular matrices for the formation of bone segments, following the colonisation of osteoblasts and subsequent calcification; of connective-dermal tissues, following the colonisation of fibroblasts), materials for the preparation of artificial skin useful in the treatment of burns or in plastic surgery; coating agents for biocompatible vascular prosthesis, carriers of pharmacologically by active ingredients in controlled-release formulations, etc.
In dermatology and cosmetology, in view of the viscoelastic and moisturising properties and of the high biocompatibility, said compositions are used both as bases for moisturising topical formulations and as invasive medical-surgical devices (xe2x80x9cfilling agentsxe2x80x9d).
The use of natural, linear hyaluronic acid for said uses is however limited by its in vivo fast degradation by enzymatic systems such as hyaluronidase, glucosidase and glucuronidase, with subsequent decrease in the molecular weight and progressive impairment of the viscoelastic properties and, generally, of the physical characteristics of the final compositions and devices (mechanical strength, elasticity, pore size,) etc.
In order to overcome this problem, mainly with the purpose of increasing the range of compositions and their applicative flexibility, chemically modified hyaluronic acids have been proposed.
Cross-linking with polyfunctional epoxides (U.S. Pat. Nos. 4,716,224, 4,772,419, 4,716,154), polyalcohols (U.S. Pat. No. 4,957,744), divinylsulphone (U.S. Pat. Nos. 4,582,865, 4,605,601, 4,636,524), aldehydes (U.S. Pat. Nos. 4,713,448, 5,128,326, 4,582,568), biscarbodiimides (U.S. Pat. No. 5,356,883), polycarboxylic acids (EP-A-718312) has been disclosed.
Said cross-linked hyaluronic acids are used as biomaterials for implants, prosthesis and medical devices, as controlled-release matrices for medicaments, as healing, anti-adhesive and dressing agents.
The sulphation of non cross-linked hyaluronic acid is generally disclosed in U.S. Pat. No. 5,013,724, mainly concerning the sulphation of heparines, heparans and dermatans for use as antithrombotic and anti-coagulant agents.
The hemisuccinylation recreation of hyaluronic acid (HY) has never been disclosed. An example of this functionalization is disclosed in EP-B-200574, claiming composite biomaterials consisting of succinylated collagen and chitosan.
The cross-linking of carboxyalkyl cellulose by means of di- or polyamines is disclosed in EP-A-566118 (Kimberly Clark Corp) for the preparation of absorbing materials with HY as cross-linking agent, by heating. Such a method appears to be economically advantageous and suitable for the large-scale productions required for this kind of products.
EP-A-462 426 (Fidia) discloses perforated biocompatible membranes and their uses as artificial skin. Collagen cross-linked with diamines and hyaluronic acid are generically cited as possible materials for said membranes.
It has now been found that new cross-linked hyaluronic acids obtainable by reaction of suitably activated carboxy groups of HY with a polyamine, as well as the salts and complexes with suitable organic or inorganic cations, have advantageous chemico-physical and biological properties for the biomedical and cosmetic uses.
The main chemico-physical and biochemical characteristics of the compounds of the invention are:
high biocompatibility;
high resistance to enzymatic degradation mainly after sulphation;
high capacity to adsorb water, with formation of visco-elastic characteristics dependent on the cross-linking degree as well as on sulphation and/or hemi-succinylation degree;
ability to chelate metal ions such as zinc or copper; said derivatives having very good stability.
The biological behaviour is new and surprising; it is known that sulphation (or supersulphation) of glycosaminoglycans such as heparin, dermatan sulphate, chondroitin and native hyaluronic acid is known to increase their anti-coagulant properties (inhibition of Xa and IIa factors and/or change of their ratio) with respect to the starting product (U.S. Pat. No. 5,013,724).
The compounds of the invention, when sulphated, have a slight anticoagulant activity, whereas it is completely surprising the lack of platelet activation and aggregation (measured as antiadhesive activity; P.R.P. model in rabbits subjected to behavioural stress, described in xe2x80x9cAbstract IL 15xe2x80x9dxe2x80x94International Conference on Advances in Biomaterials and Tissue Engineering, 14-19 Juin 1998, Capri Italy) both for the cross-linked hyaluronic acid of the invention (with different cross-linking degrees) and for the corresponding sulphate esters; this property is totally absent in the natural hyaluronic acid and esther derivatives.
No polymeric materials for medical use up to now known apparently shares the same property.